Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E
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ABSTRACT:
SUBMITTER: Phan M
PROVIDER: S-EPMC9409060 | biostudies-literature | 2022 Aug
REPOSITORIES: biostudies-literature
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Project description:Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction. Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4-5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells. Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation. Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.
| S-EPMC6491902 | biostudies-literature
Project description:Human cytomegalovirus (HCMV) infection is the most common cause of congenital viral infections and a major source of morbidity and mortality after organ transplantation. NK cells are pivotal effector cells in the innate defense against CMV. Recently, hallmarks of adaptive responses, such as memory-like features, have been recognized in NK cells. HCMV infection elicits the expansion of an NK cell subset carrying an activating receptor heterodimer, comprising CD94 and NKG2C (CD94/NKG2C), a response that resembles the clonal expansion of adaptive immune cells. Here, we determined that expansion of this NKG2C(+) subset and general NK cell recovery rely on signals derived from CD14(+) monocytes. In a coculture system, a subset of CD14(+) cells with inflammatory monocyte features produced IL-12 in response to HCMV-infected fibroblasts, and neutralization of IL-12 in this model substantially reduced CD25 upregulation and NKG2C(+) subset expansion. Finally, blockade of CD94/NKG2C on NK cells or silencing of the cognate ligand HLA-E in infected fibroblasts greatly impaired expansion of NKG2C(+) NK cells. Together, our results reveal that IL-12, CD14(+) cells, and the CD94/NKG2C/HLA-E axis are critical for the expansion of NKG2C(+) NK cells in response to HCMV infection. Moreover, strategies targeting the NKG2C(+) NK cell subset have the potential to be exploited in NK cell-based intervention strategies against viral infections and cancer.
| S-EPMC4348979 | biostudies-literature
Project description:Adaptive natural killer (NK) cell responses to human cytomegalovirus infection are characterized by the expansion of NKG2C(+) NK cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs). Here, we set out to study the HLA class I dependency of such NKG2C(+) NK cell expansions. We demonstrate the expansion of NKG2C(+) NK cells in patients with transporter associated with antigen presentation (TAP) deficiency, who express less than 10% of normal HLA class I levels. In contrast to normal individuals, expanded NKG2C(+) NK cell populations in TAP-deficient patients display a polyclonal KIR profile and remain hyporesponsive to HLA class I-negative target cells. Nonetheless, agonistic stimulation of NKG2C on NK cells from TAP-deficient patients yielded significant responses in terms of degranulation and cytokine production. Thus, while interactions with self-HLA class I molecules likely shape the KIR repertoire of expanding NKG2C(+) NK cells during adaptive NK cell responses in normal individuals, they are not a prerequisite for NKG2C(+) NK cell expansions to occur. The emergence of NKG2C-responsive adaptive NK cells in TAP-deficient patients may contribute to antiviral immunity and potentially explain these patients' low incidence of severe viral infections.
| S-EPMC4594010 | biostudies-literature
Project description:Natural killer (NK) cells play a dual role in the defense against viral pathogens by directly lysing infected cells as well as by regulating anti-viral T cell immunity. Infection by human cytomegalovirus (HCMV) promotes a persistent expansion of NKG2C+ adaptive NK cells which have been shown to display enhanced antibody-dependent responses against infected targets and associated to viral control in transplanted patients. Based on gene expression data showing increased transcription of CIITA and several genes related to the MHC class II pathway in adaptive NK cells, we explored their putative capacity for antigen presentation to CD4+ T cells. Phenotypic analysis confirmed a preferential steady-state expression of HLA-DR by circulating NKG2C+ adaptive NK cells in healthy individuals. Expression of HLA-DR in NKG2C+ adaptive NK cells was variable and unrelated to the expression of activation (i.e., CD69 and CD25) or differentiation (i.e., FcRγ chain, CD57) markers, remaining stable over time at the individual level. Incubation of purified NK cells with HCMV complexed with serum specific antibodies induced an up-regulation of surface HLA-DR concomitant to CD16 loss whereas no changes in CD80/CD86 co-stimulatory ligands were detected. In addition, surface CX3CR1 decreased upon antigen-loading while HLA-DR+ NK cells maintained a CCR7-, CXCR3low homing profile. Remarkably, HCMV-loaded purified NK cells activated autologous CD4+ T cells in an HLA-DR dependent manner. The fraction of T lymphocytes activated by antigen-loaded NK cells was smaller than that stimulated by monocyte-derived dendritic cells, corresponding to CD28-negative effector-memory CD4+ T cells with cytotoxic potential. Antigen presentation by NK cells activated a polyfunctional CD4+ T cell response characterized by degranulation (CD107a) and the secretion of Th1 cytokines (IFNγ and TNFα). Overall, our data discloses the capacity of NKG2C+ adaptive NK cells to process and present HCMV antigens to memory CD4+ cytotoxic T cells, directly regulating their response to the viral infection.
| S-EPMC6456717 | biostudies-literature
Project description:In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.
| S-EPMC9148069 | biostudies-literature
Project description:Describe a novel GMP-compliant protocol to expand clinically relevant numbers of adaptive NK cells from third-party ‘superdonors’. These NK cells provide strong reactivity in a mouse model of AML as well as against primary AML blasts ex vivo. How this study might affect research, practice or policy: These pre-clinical data demonstrate the feasibility of an NK cell-based cell therapy with a non-engineered and yet highly specific NK cell population, representing the first route to clinical testing of missing-self recognition.
2022-09-20 | E-MTAB-12228 | biostudies-arrayexpress
Project description:In this work, we analyzed the phenotype and growth of human NK cell clones obtained by the stimulation of individual NK cells with IL-2 and gene-modified K562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). We generated clones from NK cells at distinct differentiation and activation stages, determined by CD56, CD57 and HLA-DR expression levels. Less differentiated CD56bright NK cell subsets showed higher cloning efficiency compared with more differentiated CD56dim subsets, especially with the CD57bright subset. However, clones from the CD56dimCD57- subset lived longer on average than other subsets. Moreover, several clones with the highest cell numbers were derived from CD56dimCD57-HLA-DR-cells. Most of the clones including those derived from more differentiated CD56dimCD57+/-NKG2A- NK cells showed a less-differentiated NKG2A+ phenotype. Also, CD57- cells were frequently observed in clones derived from CD57+ NK cells suggesting the loss of CD57 during the cloning process. On the other hand, KIR surface expression once detected for a clone never disappeared entirely, confirming irreversibility of the KIR expression. In summary, we have demonstrated that in specific conditions terminally differentiated CD57+ human NK cells are able to acquire the CD57- phenotype that was previously not observed and, thus, was considered impossible.
| S-EPMC6281266 | biostudies-literature
Project description:Polymorphisms in the human leukocyte antigen (HLA) class I genes can cause the rejection of pluripotent stem cell (PSC)-derived products in allogeneic recipients. Disruption of the Beta-2 Microglobulin (B2M) gene eliminates surface expression of all class I molecules, but leaves the cells vulnerable to lysis by natural killer (NK) cells. Here we show that this 'missing-self' response can be prevented by forced expression of minimally polymorphic HLA-E molecules. We use adeno-associated virus (AAV)-mediated gene editing to knock in HLA-E genes at the B2M locus in human PSCs in a manner that confers inducible, regulated, surface expression of HLA-E single-chain dimers (fused to B2M) or trimers (fused to B2M and a peptide antigen), without surface expression of HLA-A, B or C. These HLA-engineered PSCs and their differentiated derivatives are not recognized as allogeneic by CD8+ T cells, do not bind anti-HLA antibodies and are resistant to NK-mediated lysis. Our approach provides a potential source of universal donor cells for applications where the differentiated derivatives lack HLA class II expression.
| S-EPMC5548598 | biostudies-literature
Project description:Adoptive natural killer (NK) cell therapy in combination with hematopoietic stem cell transplantation (HSCT) can provide cure for acute myeloid leukemia (AML) but is in part dependent on variable NK cell reactivity. Adaptive NK cells are a highly potent NK cell subsets that can be utilized to maximize ‘missing-self’ reactivity against tumor cells. What this study adds: It describes a novel GMP-compliant protocol to expand clinically relevant numbers of adaptive NK cells from third-party ‘superdonors’. These NK cells provide strong reactivity in a mouse model of AML as well as against primary AML blasts ex vivo. How this study might affect research, practice or policy: These pre-clinical data demonstrate the feasibility of an NK cell-based cell therapy with a non-engineered and yet highly specific NK cell population, representing the first route to clinical testing of missing-self recognition.
2022-09-26 | GSE214042 | GEO
Project description:BackgroundAdaptive human natural killer (NK) cells are an NK cell subpopulation arising upon cytomegalovirus (CMV) infection. They are characterized by CD94/NKG2C expression, a mature CD57+KIR+NKG2A- phenotype, a prolonged lifespan, and remarkable antitumor functions. In light of these features, adaptive NK cells represent suitable candidate to design next-generation therapies, based on their enhanced effector function which could be further boosted by Chimeric Antigen Receptors-engineering, or the combination with cell engagers. For therapeutic approaches, however, it is key to generate large numbers of functional cells.PurposeWe developed a method to efficiently expand adaptive NK cells from NK-enriched cell preparations derived from the peripheral blood of selected CMV-seropositive healthy donors. The method is based on the use of an anti-CD94 monoclonal antibody (mAb) combined with IL-2 or IL-15.ResultsBy setting this method we were able to expand high numbers of NK cells showing the typical adaptive phenotype, CD94/NKG2C+ CD94/NKG2A- CD57+, and expressing a single self-inhibitory KIR. Expanded cells maintained the CMV-induced molecular signature, exhibited high ADCC capabilities and degranulation against a HLA-E+ target. Importantly, mAb-expanded adaptive NK cells did not upregulate PD-1 or other regulatory immune checkpoints that could dampen their function.ConclusionsBy this study we provide hints to improve previous expansion methods, by eliminating the use of genetically modified cells as stimulators, and obtaining effectors not expressing unwanted inhibitory receptors. This new protocol for expanding functional adaptive NK cells is safe, cost-effective and easily implementable in a GMP context, suitable for innovative immunotherapeutic purposes.
| S-EPMC11825780 | biostudies-literature